NO146199B - PROCEDURE FOR THE PREPARATION OF ALFA-6-DEOXY-5-HYDROXYTETRACYCLINE HYDROCHLORIDE - Google Patents

Description

The present invention relates to a method for synthesizing the antibiotic α-6-deoxy-5-hydroxytetracycline via reduction of 6-methylene-5-hydroxytetracycline.

The reaction of 6-methylenetetracyclines, 11a-halo-6-methylenetetracyclines, acid addition salts thereof and polyvalent metal-salt complexes thereof with hydrogen in the presence of a heterogeneous noble metal catalyst to prepare the corresponding epimeric α- and G-6-deoxytetracyclines is described in U.S. patent no. 3 200 149. Use of a poisoned noble metal catalyst to achieve the same conversion but with an increase in the ratio between α- and 3-6-deoxytetracycline is discussed in U.S. Pat. Patent Document No. 3,444,198. U.S. Patent No. 2,984,686 describes the use of catalytic reduction using hydrogen and a noble metal catalyst to achieve only 11a-dehalogenation of 11a-halo-6-methylenetetracyclines. Moreover, reduction of 11a-halo-6-methylenetetracyclines, salts and complexes thereof in the presence of a noble metal catalyst using hydrazines as a hydrogen source is discussed in German patent document No. 2,131,944. British Patent No. 1,296,340 describes the use of Raney nickel and Raney cobalt as catalysts for such reductions.

Rhodium halide complexes containing tertiary phosphine or arsine ligands, their preparation and use as homogeneous hydrogenation catalysts are described in U.S. Pat. Patent Document No. 3,639,439, issued February 1, 1972. Soluble complexes of metals from the platinum group, especially of rhodium, containing a halide and a tertiary phosphine, arsine, stibine or amine, their preparation and use as hydrogenation catalysts are also disclosed in British Patent Nos. 1,138,601 (published January 1, 1969), No. 1,219,763 (published January 20, 1971), No. 1,121,642

(published July 31, 1968) and No. 1,121,643 (published July 31, 1968), and in U.S. Pat. Patents No. 3,489 (January 13, 1970) and No. 3,549,780 (August 5, 1969). Such catalysts are indicated to provide an improved process for the hydrogenation of unsaturated organic compounds, especially olefins, compared to the use of heterogeneous catalysts.

German off. document no, 2,308,227, published on 30 August 1973, describes the production of α-6-deoxytetracyclines by homogeneous catalytic hydrogenation using tris(triphenylphosphine)chlororhodium as catalyst. It is stated that the catalyst can be formed in advance or prepared directly in the reaction medium by dissolving rhodium trichloride in the medium in the presence of between one and three molar equivalents of triphenylphosphine.

U.S. patent no. 3,692,864, issued on 19 September 1972, mentions the hydrogenation of unsaturated organic molecules using homogeneous metal complexes of the iron-triad type (nickel, cobalt, iron) together with tertiary phosphines. A typical example of the described complexes is chlorotris(triphenylphosphine)cobalt(I)„

Numerous publications mention homogeneous catalysis as promising for hydrogenation reactions, including regiospecific, selective and asymmetric reductions. Knowles et al., Chem. Commun., p. 1445 (1968), Horner et al., Angew. Chem., Int. Ed., 1_,

942 (1968) and Belgian Patent No. 766,960, published November 10, 1971, refer to the use of complexes of monovalent rhodium together with optically active tertiary phosphine ligands as homogeneous catalysts to achieve asymmetric catalytic hydrogenation. Later publications give a fairly comprehensive review of the state of the art. Harmon et al., Chem. Rev., 73, 21-52 (1973), Knowles et al., Chem. Commun. , p. 10 (1972), Grubbs et al., J. Am. Chem. Soc, 93_, 3062 (1971), Kagan et al., J. Am, Chem. Soc, 94, 6929 (1972) and "Homogeneous Catalysis, Industrial Applications and Implications", vol. 70, Advances in Chemistry Series, published by the American Chemical Society, Washington, D.C. (1968), "Aspects of Homogeneous Catalysis", vol. I, pp. 5-75 (1970), edited by R. Ugo and published by Carlo Manfredi, Milan, Italy and Vol'Pin et al., Russian Chemical Reviews, 38, 273-289 (1969).

Homogeneous catalytic hydrogenation of exocyclic methylene groups in methylenecyclohexanes (Augustine et al., Ann. N.Y. Sei., 158, 482-91 [1969]), coronopilin (Ruesch et al., Tetrahedron, 25_, 807-11 [1969], and in an intermediate in the stereoselective total synthesis of Seychelles (Piers et al., chem. Communs. 1069-70

[1969]) using tris(triphenylphosphine)chlororhodium as catalyst, is described.

In all of the preceding mention of the state of the art, the reduction of a substance is carried out using hydrogen gas in the presence of a reduction catalyst.

Fichtmann et al., J. Org. Chem., 33_, 1281 (1968) and Taylor et al., J. Org. chem., 3_7_, 3913 (1972) mentions cobalt hydrocarbonyl, HCo(C0)3 or HCo(C0)4, as substances capable of reducing olefins or aromatic substrates, the said cobalt hydrocarbon being formed by reduction of dicobalt octacarbonyl by using hydrogen gas at elevated temperatures.

A new process has now been found for the production of α-6-deoxy-5-hydroxy-tetracycline hydrochloride which has the formula

by reduction of 6-methylene-5-hydroxytetracycline with the formula

or the hydrochloride salt thereof in the presence of a catalyst.

According to the invention, 6-methylene-5-hydroxytetracycline or its hydrochloride salt is brought into contact with dicobolt octacarbonyl, triphenylphosphine and mineral acid at a molar ratio between 6-methylene-5-hydroxytetracycline/dicobolt octacarbonyl/triphenylphosphine/mineral acid of 1.0/1.0 /0.1-0.2/1.5-3.5 in a solvent for said reactants under an inert atmosphere and a reaction temperature of 80-115°C.

Completely unexpected in the method according to the invention, is that the reduction of the exocyclic double bond in the 6-methylenetetracycline proceeds so easily without the need for hydrogen gas either in the reaction itself or in the formation of the reaction reagents, and also the discovery that the tetracycline molecule is not destroyed or forms irreversible complexes with the reagents Co2(C0)g, hydrochloric acid and/or triphenylphosphine.

An equally unexpected discovery that makes the present method of commercial interest is the highly specific nature of the reduction, which results in almost exclusive formation of the antibiotic α-6-deoxy-5-hydroxytetracycline as opposed to the corresponding B-epimer.

The aforementioned reduction reaction is shown in the following diagram:

where HA means a mineral acid, hydrochloric acid.

The reaction is carried out in a reaction dissolution medium1. It is preferred that said solvent is of such a nature that all the reactants are kept dissolved under the reaction conditions of the method. Towards the end of this, it is preferred to use a solvent for dissolving the dicobolt octacarbonyl and the mineral acid addition salt of the 6-methylenetetracycline. The most convenient solvent for this purpose is a mixture of a liquid aromatic solvent, such as benzene, toluene, or xylene, and an aprotic, highly polar solvent, such as dimethylformamide, dimethylacetamide, or hexamethylphosphoramide. The aromatic solvents serve as excellent solvents for keeping the cobalt carbonyl in solution, and polar aprotic solvents serve a similar role for the 6-methylenetetracycline, the triphenylphosphine, and the mineral acid. When using these solvents for the present method, it is preferred that only a minimal amount of said solvents is used which is just sufficient to dissolve the reactants used. Use of relatively larger amounts of solvents will, as a person skilled in the art will readily understand, lead to longer reaction times and give rise to cleavage of either reactants or the products formed.

The preferred solvents for the method according to the present invention are benzene-dimethylformamide and benzene-dimethylacetamide.

As previously mentioned, the atmosphere above the reaction surface should be of an inert nature, and not react to a noticeable extent with either starting materials or products. Such gases as oxygen, air or carbon dioxide have a noticeable harmful effect on a favorable formation of the desired products. Accordingly, the atmosphere is suitably constituted by such gases as nitrogen, argon or helium, with nitrogen and argon being preferred.

Although the temperature range for the reaction in which this reduction can be carried out is relatively wide, being from 80-115°C, exceeding these limits has a detrimental effect on the result of said reaction. Reaction temperatures below 70°C lead to very slow reduction and often to incomplete conversion, even after prolonged reaction times, while reaction temperatures above 115°C cause extensive cleavage of the desired α-6-deoxy-5-hydroxytetracycline product. The preferred reaction temperature at which complete formation of the desired product is achieved without

extensive cleavage of said product, is from about 90-95°c.

v As one skilled in the industry will readily understand, the reaction time is dependent on a number of reaction variables, including reaction temperature, intrinsic reactivity in the starting materials and concentration of the reactants in the solvent medium. By judicious use of only sufficient solvent to dissolve all starting reagents, as previously mentioned, and by using a temperature range of 90-95°C, the reaction process according to the present invention is completed in about 4 hours.

A necessary feature of the present method is the use of a mineral acid. For this purpose, hydrochloric acid seems to give optimal results, although, as a person skilled in the industry can easily understand, other mineral acids can also be used, such as hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfuric acid, phosphoric acid or nitric acid. The 6-methylene-5-hydroxytetracycline starting material can be added as free base and the hydrochloric acid can be added separately to the reaction mixture, or alternatively the starting tetracycline can be added as a hydrochloric acid addition salt and further hydrochloric acid can be added if necessary. Due to the poor storability of the starting tetracycline as a free base and the stability and availability of the hydrochloride salt, the latter is preferred as the starting tetracycline.

Under reaction conditions where only one flour or one equivalent of hydrochloric acid is used, the desired α-6-deoxy-5-hydroxytetracycline is formed as the hydrochloride salt, but not in optimal yield or purity. It is therefore preferred to use more than molar or equivalent amounts of acid in relation to 6-methylenetetracycline, and to achieve this one can consequently add additional acid to the reaction mixture. Since the α-6-deoxy-5-hydroxytetracycline product is not stable under the reaction conditions of the present method in the presence of too high an acid concentration, no more than 3.5 equivalents of acid are used per

mol of tetracycline, and it is further preferred that the amount of hydrochloric acid used is 1.5-2 equivalents per moles of tetra-cycline starting material le.

Of the aforementioned mineral acids that can be used in the present process, hydrochloric acid is the preferred mineral acid. In terms of experience, it is often convenient, when hydrochloric acid is used, to

adding the hydrochloric acid or another mineral acid to a solution of the commercially available 6-methylene-5-hydroxytetracycline hydrochloride,

dissolved in a polar, aprotic solvent until the desired amount of acid has been added.

Another of the starting reagents that has a profound effect

on the product formation according to the present method, is triphenylphosphine. Other phosphines have been used, including trialkyl, tricycloalkyl, and substituted triphenyl phosphines, but without the success associated with triphenylphosphine. Furthermore, the amount of this reagent used has a marked effect on the ratio between α-6-deoxy-5-hydroxytetracycline, which is the preferred product in the present process, and β-6-deoxy-5-hydroxytetracycline. A ratio of 0.1-0.2 equivalents of triphenylphosphine per moles of 6-methylenetetracycline is useful to provide an advantageous ratio between the α- and β-6-deoxy-5-hydroxytetracycline products, and a preferred ratio is 0.1 equivalents of triphenylphosphine per moles of tetracycline starting material.

With respect to the amount of dicobolt octacarbonyl used in the present process, it has been found that the use of as much as 2 to 5 equivalents per mol of 6-methylenetetracycline does not give any advantages over the use of an equivalent with regard to the total reduction and to the distribution of α- and β-6-deoxy-5-hydrotetracycline in the product. Use of from less than one equivalent to as little as 0.1 equivalents causes a continuous marked reduction in the total amount of product formed. For economic reasons, it is preferred that one equivalent of dicobolt octacarbonyl is used per moles of starting tetracycline.

The order in which the reactants are mixed in the present process is not critical, although a slightly higher reduction yield is achieved by using a process where the reactants dicoboltoctacarbonyl, triphenylphosphine and hydrochloric acid and the aprotic, polar solvent are stirred at 85°C for approximately 10 minutes before the addition of the 6-methylenetetracycline or the hydrochloride salt.

When examining the range of the reaction variables according to the present method, it is extremely time-consuming to carry out experiments with the reaction mixture by isolating and identifying. the reduction products, and consequently economical samples such as thin-layer chromatography and high-pressure liquid chromatography are often used.

At the end of the reaction, the α-6-deoxy-5-hydroxytetracycline product is present in the reaction mixture as the hydrochloric acid addition salt. With regard to the isolation of the α-6-deoxy-5-hydroxy-tetracycline from the reaction mixture, it is preferred that the mixture be filtered and the aromatic solvent removed under reduced pressure. The remaining polar solvent is then filtered and treated with a saturated aqueous solution of sulfosalicylic acid. The subsequent addition of water precipitates the sulpho-salicylic acid salts of the reduction products and also any unreduced 6-methylenetetracycline. Further purification is carried out by recrystallization from methanol-water.

In the preferred reaction conditions for the reduction of 6-methylene-5-hydroxytetracycline to α-6-deoxy-5-hydroxytetracycline, the above-mentioned tetracycline, dicobolt octacarbonyl and triphenylphosphine with a molar ratio of 1.0/1.0 /0.1 brought into contact with hydrochloric acid in a solvent of benzene-dimethylacetamide or benzene-dimethylformamide in an inert atmosphere of nitrogen or argon at a reaction temperature of 90-95°C.

The chemistry of tetracyclines and their use as antibiotic substances when mixing different microbial infections is well known and extensively documented in the medical literature. Much of the background for these agents, including α-6-deoxy-5-hydroxytetracycline, and how they are used as therapeutic agents, is discussed by R.K. Blackwood, Encyclopedia of Chemical Technology, 20, 1-33, (1969). Measurements with regard to the presence of the α-isomer and the amount that is present are therefore the only thing necessary to prove the effect of the method.

The following examples are given to illustrate the invention,

and many variations of these are possible within the spirit and scope of the invention.

EXAMPLE 1

To 5.5 mg (0.021 mmol) of triphenylphosphine in 5 ml of dry benzene, which is in a closed three-necked flask under a nitrogen atmosphere, via a syringe through a rubber diaphragm covering a neck opening, is added 360 mg (1.05 mmol) of dicobolt octacarbonyl in 10 ml of the same solvent. After stirring for 30 minutes at room temperature, 100 mg (0.21 mmol) of 6-methylene-5-hydroxytetracycline hydrochloride in 10 ml of dimethylformamide is injected into the reaction flask, and the resulting solution is heated to 70°C.

for 18 hours.

A sample of the reaction mixture is removed and chromatographed on a silica gel plate which has been previously treated with a syringe solution of 0.005 N sodium acetate and 0.002 N ethylenediaminetetraacetic acid adjusted to a pH value of 6 with acetic acid and then dried at 110° C overnight. The chromatography is carried out in an ascending system of 95% tetrahydrofuran and is developed with ammonia. α-Deoxy-5-hydroxytetracycline appears as a yellow spot under a 366 nyu ultraviolet lamp. The results show a ratio between α-6-deoxy and β-6-deoxy-5-hydroxy-tetracycline of 5 to 1.

The remainder of the reaction mixture is filtered and the benzene is removed under reduced pressure. The remaining green solution is filtered and the filtrate is treated first with 2 ml of a 10% aqueous solution of sulfosalicylic acid and then with 6 ml of water. The resulting slurry is, after stirring for a few minutes, filtered and the solids are washed with water and dried.

The solids are dissolved in a minimal amount of water and applied to a high-pressure liquid chromatography column packed with a quaternary ammonium-substituted methacrylate polymer coated with 1%

on a surface-controlled porosity support (U.S. Patent Nos. 3,485,658 and 3,505,785). The product is eluted with a buffer of 0.005 n sodium acetate and 0.002 n ethylenediaminetetraacetic acid adjusted to a pH value of 5.8 with acetic acid, under a pressure of 119 kg/cm . An ultraviolet observer (254 iryu) at the column exit measures the tetracycline in the eluate. Ultraviolet measurement is, in turn, recorded with a variant A-25 pin meter. Measurements show that the yield of α-6-deoxy- and 6-6-deoxy-5-hydroxy-tetracycline is 35-40%, of which 83% is the desired α-epimer and 17%

is the B epimer.

EXAMPLE 2

In a similar method as in example 1,

72 mg (0.21 mmol) of dicobolt octacarbonyl in 5 mL of benzene was added to 5.5 mg (0.021 mmol) of triphenylphosphine in 5 mL of benzene under a nitrogen atmosphere, followed by lOO mg (0.21 mmol) of 6-methylene-5- hydroxytetracycline hydrochloride in 10 ml of dimethylformamide, and the mixture is kept under a nitrogen atmosphere. To the resulting reaction mixture is added 1 ml of benzene containing 19.6 mg (0.21 mmol) of sulfuric acid, and the mixture is heated to 70°C for 16 hours. Experiments with high-pressure liquid chromatography of the reaction mixture, carried out as in example 1, show that the reduction provides a-6-

deoxy-5-hydroxytetracycline.

EXAMPLE 3

Substantially similar results are obtained when phosphoric acid, hydrobromic acid, hydroiodic acid and nitric acid are used instead of sulfuric acid in the process of Example 2.

EXAMPLE 4

To a three-necked flask filled with argon gas are added 35 mg (0.13 mmol) of triphenylphosphine in 0.5 ml of benzene and 180 mg (0.52 mmol) of dicobolt octacarbonyl in 4.5 ml of benzene, and the resulting solution is left to stir at room temperature for 15 minutes. A solution of 250 mg (0.52 mmol) of 6-methylene-5-hydroxy-tetracycline hydrochloride in 2.5 ml of dimethylacetamide is added, followed by 0.6 ml of a 1 : 1 (vol./vol.) soln. of dimethylacetamide-12 n hydrochloric acid (3.4 mmol), and the reaction mixture is heated to 115°C for 4 hours.

After the heating period, the mixture is allowed to cool and is filtered. The filtrate is evaporated under reduced pressure to remove the benzene, and is again filtered. A solution of 5.0 ml of a 10% aqueous sulfosalicylic acid is added to the filtrate, followed by 15 ml of water. The precipitated sulfosalicylic acid salt is filtered and dried, 445 mg (72% yield). Experiments with high-pressure liquid chromatography on a sample of the salts, in accordance with example 1, show that the product consists of 75-80% a-6-deoxy-5-hydroxy-tetracycline, 1-2% B- 6-deoxy-5-hydroxytetracycline and 15-20% unreduced 6-methylene-5-hydroxytetracycline.

The crude product is recrystallized from methanol-water and is converted to the hydrochloride salt by addition of an ethanol-water solution containing hydrochloric acid. The precipitated hydrochloride salt is further purified by recrystallization from ethanol containing a minimal amount of water.

EXAMPLE 5

To 27.5 mg (0.104 mmol) of triphenylphosphine in 5 ml of benzene under a nitrogen atmosphere are added 180 mg (0.52 mmol) of dicobolt octacarbonyl in 5 ml of the same solvent, 250 mg of 6-methylene-5-hydroxytetracycline hydrochloride in 5 ml of dimethylformamide and 18 mg (0.52 mmol) of hydrogen chloride in 1 ml of dimethylformamide, and the resulting reaction mixture is heated at 85°C for 3 hours. High pressure liquid chromatography shows that there is no remaining tetracycline starting material and that the reduction is complete.

The reaction mixture is cooled to room temperature and is worked up as described in example 1. The sulfosalicylic acid salts, 260 mg, consist of 98% α-6-deoxy-5-hydroxytetracycline. The unprocessed sulfosalicylic acid salts are converted into hydrochloride salts by adding said salts to ethanol-water containing hydrochloric acid. Further recrystallization from methanol-water provides pure α-6-deoxy-5-hydroxytetracycline hydrochloride.

EXAMPLE 6

The procedure from example 5 is repeated with the exception that the indicated phosphines are used instead of triphenylphosphine. Yields are from a-6-deoxy-5-hydroxytetracycline (a-doxy), B-6-deoxy-5-hydroxytetracycline (B-doxy) and the starting material 6-methylene-5-hydroxytetracycline (metacycline).

EXAMPLE 7

starting from 27.5 mg (0.1 mmol) triphenylphosphine in 25 ml benzene, 342 mg (1 mmol) dicoboltoctacarbonyl in 10 ml benzene and 399 mg (1 mmol) 6-methylene-5-hydroxytetracycline in 40 ml dimethyl form amide containing 54 mg (1.5 mmol) of hydrogen chloride, the procedure of Example 1 is repeated to give α-deoxy-5r-hydroxytetracycline in comparable yields.

Claims (6)

1. Process for the preparation of α-6-deoxy-5-hydroxy-tetracycline hydrochloride with the formula by reduction of 6-methylene-5-hydroxy-tetracycline with the formula or the hydrochloride salt thereof in the presence of a catalyst, characterized in that 6-methylene-5-hydroxytetracycline or the hydrochloride salt is brought into contact with dicobolt octacarbonyl, triphenylphosphine and mineral acid with a molar ratio of 6-methylene-5-hydroxytetracycline/dicobolt octacarbonyl/triphenylphosphine/mineral acid of 1.0/1.0/0.1-0.2/1.5-3.5 in a solvent for the reactants and under an inert atmosphere at a reaction temperature of 80-115°C. 2. Method according to claim 1, characterized in that hydrochloric acid is used as the mineral acid. 3. Method according to any of the requirements log 2, characterized in that a ratio of 1.0/1.0/0.1-0.2/1.5-2 is used. 4. Method according to any one of claims 1 to 3, characterized in that the reaction temperature is kept between 90 and 95°C. 5. Method according to any one of claims 1 to 4, characterized in that a molar ratio of 1.0/1.0/0.1/1.5 is used. 6. Method according to any one of claims 1 to 5, characterized in that benzene-dimethylformamide or benzene-dimethyllacetajnide is used as solvent.